1. Field of the Invention
The present invention relates to the field of molecular biology and protein biochemistry. More particularly, it relates to purification and characterization of proteins present in the matrix of cartilage-containing tissues, and to the cloning and characterization of genes encoding these proteins.
2. Description of the Related Art
Articular cartilage is a heterogeneous tissue in which cells are arranged in layers, forming a matrix. The layers have a different composition and function depending on their location with respect to the articular surface of the subchondral bone. The extracellular matrix is also arranged into compartments around the cells: pericellular (closest to the cell), territorial (extending around individual or groups of chondrocytes), and interterritorial matrix (farthest away from the cells). The matrix is produced by the chondrocytes and contains, as major constituents, fibril-forming DERSON, collagens and large aggregating proteoglycans that are assembled into highly organized structures (Heinegård, D. and Oldberg, Å., 1993, In Connective Tissue and Its Hereditable Disorders (Royce, P. M. and Steinmann, B., eds.) pp. 103–147, Wiley-Liss Inc., New York). Collagen confers tensile properties to the tissue, whereas proteoglycans play a key role in the normal resilience and load dissipation of the cartilage.
There is also a minor population of non-collagenous proteins for which no functional role has yet been identified. They might have roles in maintaining the tissue homeostasis by the regulation of matrix assembly, cell recognition, and cell attachment. They might also have a part to play in balancing the processes of cartilage repair and degradation, as well as in disease processes where degradation outbalances repair, and loss of tissue ensues. This third set of matrix proteins are the non-collagenous glycoproteins, including several members of the family of leucine-rich repeat (LRR) proteins and the thrombospondins. The LRR proteins include decorin, biglycan, fibromodulin, and lumican, all with the capacity to bind to collagen (for refs. see Heinegård et al., 1998, “Biochemistry and metabolism of normal and OA cartilage”, In Osteoarthritis, K. D. Brant, M. Doherty, and S. Lohmander, eds., Oxford University Press, New York, In press). These molecules are found along surfaces of collagen fibrils in the tissue.
Other members of this family include chondroadherin (Neame et al., 1994, “The structure of a 38-kDa leucine-rich protein (Chondroaherin) isolated from bovine cartilage”, J. Biol. Chem. 269:21547–21554) and PRELP (Bengtsson et al., 1995, “The primary structure of a basic leucine-rich repeat protein, PRELP, found in connective tissues”, J. Biol. Chem. 270:25639–25644). The thrombospondin family includes the pentameric COMP, which is one of the more abundant cartilage matrix proteins (Hedbom et al., 1992, “Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage”, J. Biol. Chem. 267:6132–6136; Oldberg et al., 1992, “COMP (cartilage oligomeric matrix protein) is structurally related to thrombospondins”, J. Biol. Chem. 267:22346–22350). Furthermore, thrombospondins-1 has been identified in articular cartilage (Miller and McDevitt, 1988, “Thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes”, Biochem. Biophys. Res. Commun. 153:708–714). There are also a number of other proteins that might not be part of the described families.
Interestingly, in joint disease, such as osteoarthrosis (OA; also known as osteoarthritis) there are major alterations in the composition of the cartilage extracellular matrix. These changes reflect an altered homeostasis, and might play a role in defective repair (Heinegård et al., supra).
Preliminary studies by the present inventors and their colleagues have shown that there are a set of proteins whose synthesis and content are increased in early OA cartilage. In most cases the structure and function of these proteins are not known. However, detailed knowledge of their structure, function, and variability in normal conditions, as well as in disease, is of fundamental importance in defining mechanisms and events occurring in joint disease. Thus, to identify matrix constituents involved in these processes, it is imperative to identify, isolate, and characterize these matrix proteins.
Osteoarthritis is a non-inflammatory bone disorder characterized by a slowly progressing matrix degeneration punctuated by short periods of inflammation. During the degeneration process, there is degradation of matrix molecules, including collagens and proteoglycans and subsequent release of matrix fragments into the synovial fluid. Loss of movement and strength, in the affected joints, often due to pain, are the predominant clinical manifestations of the disease. Pain killers are the most often prescribed treatment. Because osteoarthritis is a slowly progressing disease, it is desirable to detect the disease early in its development and treat it before extensive damage has occurred. There exists a need for methods and compositions for early detection of this clinically important disease. Possible markers for osteoarthritis, which could be used early in the development of the disease, include the matrix proteins.